1. Counting cells using a haemocytometer
Kate Andrews
Lorraine Bruce

2. Cells and Proteins (Advanced Higher)
(f) Aseptic technique and cell culture
Use of inoculum, explants or cells. Use of: haemocytometers to estimate total cell counts; vital staining to estimate viable cell counts. Complex media containing growth factors from serum for animal cell lines. Lifetime of primary cell lines and cancer cell lines in culture. Use of growth regulators in plant tissue culture.
Sterilisation of containers, equipment and materials. Disinfection of working area.
Culture bacterial, yeast and algal cells using aseptic technique.
Use a haemocytometer to make an estimate of cell count.

3. Yeast cells x100

4. Saccharomyces cerevisiae as seen using an electron microscope ~ x 500,000
Image: Society for General Microbiology

5. Blood counts: for patients with abnormal blood cells, where automated counters don't perform well.
Sperm counts
Cell culture: when subculturing or recording cell population growth over time.
Beer brewing: for the preparation of the yeast cultures.
Cell processing for downstream analysis: accurate cell numbers are needed in many tests (PCR, flow cytometry) while some others require high cell viability.
Measurement of cell size: in a micrograph, the real cell size can be inferred by scaling it to the width of a haemocytometer square, which is known.

6.
Home / Biology Home / Resources / Microbiological Techniques / Enumerating micro-organisms / Counting cells using a haemocytometer

7. See SSERC website:

8. C-Chip Haemocytometer
Cheaper!
Disposable
No need for coverslips!
Virtually non-breakable!
Reusable (within limitations)

9. Image: www.invitrogen.com

11. The grid has 25 squares, we will sample 5 squares
1mm
0.2mm
The grid has 25 squares, we will sample 5 squares

12. C-Chip grid view x40

13. C-Chip grid view x100

14. Boundary lines
Top left square x400

15. Bottom right square x400

16. Loading the haemocytometer

17. Yeast cells in grid x100

18. Yeast cells in one square of the grid x400

19. Systematic counting....

20. What should be counted?

22. Volume in sample area (5 squares)
Length of side of grid = 1 mm
Area of grid = 1 mm2
Depth between coverslip
and slide = mm
Volume under grid = 1 mm2 x 0.1 mm
(25 squares)
= mm3
Volume under 5 squares = / 5 mm3
= mm3
Estimating cell concentration (in 5 squares)
Number of cells in sample = n
Number of cells in 1 mm3 = n x 50 (because 0.02 mm3 is 1/50 of 1 mm3)
Number of cells in 1 cm3 = (n x 50) x 1000

23. If the concentration of cells is very low, all the cells in the 25 square grid could be counted.
Volume in grid (25 squares)
Length of side of grid = 1 mm
Area of grid = 1 mm2
Depth between coverslip
and slide = mm
Volume under grid = 1 mm2 x 0.1 mm
(25 squares)
= mm3
Estimating cell concentration
Number of cells in grid = n
Number of cells in 1 mm3 = n x 10
Number of cells in 1 cm = (n x 10) x 1000

24. For counting large cells, some protocols advocate counting the cells in the ‘big’ squares...
Some protocols suggest averaging the number of cells counted prior to doing the calculation of cell concentration

25. Yeast cells (x 400) stained with methylene blue
In some situations cell viability is also taken into consideration –
Trypan blue
Methylene blue
Yeast cells (x 400) stained with methylene blue
Live cells actively pump out the dye.
Non-viable cells are dark blue.
Image:

26. Cell counting apps iPhone HemocyTap £1.49

27. Counting cells National 4 Cell Biology
5. Properties of microorganisms and use in industries
Properties include rapid growth, diverse use of food sources
Life on Earth
4. Fertiliser design and impact of fertilisers
Nitrogen in fresh water increases algal growth, blocking out light.
Investigate the effects of fertilisers e.g. algal blooms

28. Counting cells National 5 Cell Biology 3. Producing new cells
Appropriate growth media include various nutrient broths and agars
Carry out numeracy activities based on cell growth graphs/curves
Life on Earth
2c. Nitrogen in ecosystems
5. Impact on the environment
Fertilisers supply nitrates to increase yield
Fertilisers can leach into fresh water, increasing algal blooms. This leads to a reduction in oxygen levels

29. Counting cells Higher Biology Metabolism and survival
3. Metabolism in microorganisms –
microorganisms to include archaea, bacteria and some species of eukaryota
Environmental control of metabolism
Phases of growth and doubling or generation time of exponential growth and changes in culture conditions
Interpretation of exponential growth on normal and semi-logarithmic scales.